Near-infrared dioxetane luminophores with direct chemiluminescence emission mode

Chemiluminescent luminophores are considered as one of the most sensitive families of probes for detection and imaging applications. Due to their high signal-to-noise ratios, luminophores with near-infrared (NIR) emission are particularly important for in vivo use. In addition, light with such long wavelength has significantly greater capability for penetration through organic tissue. So far, only a few reports have described the use of chemiluminescence systems for in vivo imaging. Such systems are always based on an energy-transfer process from a chemiluminescent precursor to a nearby emissive fluorescent dye. Here, we describe the development of the first chemiluminescent luminophores with a direct mode of NIR light emission that are suitable for use under physiological conditions. Our strategy is based on incorporation of a substituent with an extended π-electron system on the excited species obtained during the chemiexcitation pathway of Schaap’s adamantylidene-dioxetane probe. In this manner, we designed and synthesized two new luminophores with direct light emission wavelength in the NIR region. Masking of the luminophores with analyte-responsive groups has resulted in turn-ON probes for detection and imaging of β-galactosidase and hydrogen peroxide. The probes’ ability to image their corresponding analyte/enzyme was effectively demonstrated in vitro for β-galactosidase activity and in vivo in a mouse model of inflammation. We anticipate that our strategy for obtaining NIR luminophores will open new doors for further exploration of complex biomolecular systems using non-invasive intravital chemiluminescence imaging techniques.

Introduction

Optical imaging modalities have become powerful tools for non-invasive visualization of biomolecular processes in real time with high spatial resolution. (1, 2) Moreover, such imaging devices are relatively inexpensive, easy to use, portable, and adaptable to acquire physiological and functional information from microscopic to macroscopic levels. (3, 4) Fluorescence is a common useful modality for optical imaging that is broadly used for detection and monitoring of various biological processes in vitro and in vivo. (5-8) The obtained signal-to-noise ratio in such measurements is often limited by light interference resulting from excitation and the autofluorescence of biological tissues. In bioluminescence imaging, such limitations are avoided, as no excitation light source is required and background signal is not affected by phenomena like autofluorescence. (9-14) In addition, bioluminescence imaging techniques rely heavily on cells that are engineered to express the enzyme luciferase.

Chemiluminescence imaging could offer some attractive advantages over bioluminescence, as light generation is initiated by a specific chemical reaction without further enzymatic dependency. (15, 16) So far, only a few reports have described chemiluminescence systems that were used for in vivo imaging. (17-29) Such systems are usually based on an energy-transfer process from a chemiluminescent precursor to a nearby emissive fluorescent dye. Furthermore, the activation mechanism of the chemiluminescent precursors used in most systems is limited by an oxidation step, which diminishes the generality of the platform.

Schaap’s adamantylidene-1,2-dioxetane (30-32) is a chemiluminescent compound that does not require a prior oxidation step, since it is already oxidized in the form of a thermally stable dioxetane (Figure 1A).

Its chemiexcitation mechanism relies on a phenol deprotection and an electron transfer from a phenolate to the peroxide bond of the dioxetane. As a result, various analyte-responsive groups can be applied to trigger the chemiexcitation mechanism. Thus, different probes can be designed for detection of a variety of analytes.

Tagged 2017